The dark side of the wool? From wool wastes to keratin microfilaments through the solution blow spinning process.

The valorization of discarded wool from dairy sheep breeding is a challenging issue. The most proposed strategies lie in the processing of keratin extracted from wool without reducing the molecular weight of the protein chains (the high molecular weight-HMW keratin). Here, the HMW keratin has been spun for the first time by solution blow spinning. A screening study of the process carried out with a 2-level full factorial design revealed that keratin filaments can be obtained by using the polyethylene oxide at 900 kDa, a 2 bar air pressure, and a 30 cm needle-collector distance. An annealing at 80 °C for 15 min, at pH 3.5 with citric acid contributes to increasing the viscosity of the keratin solutions thereby allowing the production of defect-free and water-stable filaments having diameters from 1 to 6 µm. A negligible toxic effect was observed after 24 and 48 h on HT29 epithelial cells and normal blood cells displayed behavior similar to the control demonstrating that the patches are hemocompatible. Therefore, the developed SBS process of keratin aqueous solutions could represent a valuable platform for developing patches that need to be blood-contacting and deposited in-situ.

Leveraging 3D-printed microfluidic micromixers for the continuous manufacture of melatonin loaded SNEDDS with enhanced antioxidant activity and skin permeability.

Vesicants are chemical warfare agents (CWAs) capable of causing severe skin damage and systemic toxicity. Melatonin, known for its anti-inflammatory and antioxidant properties, can mitigate the effects of these agents. Self-nano-emulsifying drug delivery systems (SNEDDS) containing a high melatonin concentration (5 %, 50 mg/g) were optimized using a quality-by-design approach from biocompatible, non-irritant excipients with a particle size of about 100 nm. The melatonin-loaded SNEDDS showed a 43-fold greater permeability than a conventional melatonin cream. Chemical stability at ambient temperature (25 °C) was maintained for one year. The preparation of optimised melatonin-loaded SNEDDS using a simple mixing method was compared to microfluidic micromixers. Mixing was successfully achieved using a 3D-printed (fused deposition modeling or stereolithography) T-shaped toroidal microfluidic chip (with a channel geometry optimized by computational fluid dynamics), resulting in a scalable, continuous process for the first time with a substantial reduction in preparation time compared to other conventional mixing approaches. No statistically significant differences were observed in the key quality attributes, such as particle size and melatonin loading, between mixing method till kinetic equilibrium solubility is reached and mixing using the 3D-printed micromixers. This scalable, continuous, cost-effective approach improves the overall efficiency of SNEDDS production, reduces the cost of quality control for multiple batches, and demonstrates the potential of continuous microfluidic manufacture with readily customizable 3D-printed micromixers at points of care, such as military bases.

Efficient and highly reproducible production of red blood cell-derived extracellular vesicle mimetics for the loading and delivery of RNA molecules.

Extracellular vesicles (EVs) are promising natural nanocarriers for the delivery of therapeutic agents. As with any other kind of cell, red blood cells (RBCs) produce a limited number of EVs under physiological and pathological conditions. Thus, RBC-derived extracellular vesicles (RBCEVs) have been recently suggested as next-generation delivery systems for therapeutic purposes. In this paper, we show that thanks to their unique biological and physicochemical features, RBCs can be efficiently pre-loaded with several kinds of molecules and further used to generate RBCEVs. A physical vesiculation method, based on "soft extrusion", was developed, producing an extremely high yield of cargo-loaded RBCEV mimetics. The RBCEVs population has been deeply characterized according to the new guidelines MISEV2023, showing great homogeneity in terms of size, biological features, membrane architecture and cargo. In vitro preliminary results demonstrated that RBCEVs are abundantly internalized by cells and exert peculiar biological effects. Indeed, efficient loading and delivery of miR-210 by RBCEVs to HUVEC has been proven, as well as the inhibition of a known mRNA target. Of note, the bench-scale process can be scaled-up and translated into clinics. In conclusion, this investigation could open the way to a new biomimetic platform for RNA-based therapies and/or other therapeutic cargoes useful in several diseases.

Biomimetic Platelet-Cloaked Nanoparticles for the Delivery of Anti-Inflammatory Curcumin in the Treatment of Atherosclerosis.

Atherosclerosis still represents a major driver of cardiovascular diseases worldwide. Together with accumulation of lipids in the plaque, inflammation is recognized as one of the key players in the formation and development of atherosclerotic plaque. Systemic anti-inflammatory treatments are successful in reducing the disease burden, but are correlated with severe side effects, underlining the need for targeted formulations. In this work, curcumin is chosen as the anti-inflammatory payload model and further loaded in lignin-based nanoparticles (NPs). The NPs are then coated with a tannic acid (TA)- Fe (III) complex and further cloaked with fragments derived from platelet cell membrane, yielding NPs with homogenous size. The two coatings increase the interaction between the NPs and cells, both endothelial and macrophages, in steady state or inflamed status. Furthermore, NPs are cytocompatible toward endothelial, smooth muscle and immune cells, while not inducing immune activation. The anti-inflammatory efficacy is demonstrated in endothelial cells by real-time quantitative polymerase chain reaction and ELISA assay where curcumin-loaded NPs decrease the expression of Nf-κb, TGF-ß1, IL-6, and IL-1ß in lipopolysaccharide-inflamed cells. Overall, due to the increase in the cell-NP interactions and the anti-inflammatory efficacy, these NPs represent potential candidates for the targeted anti-inflammatory treatment of atherosclerosis.